Telomere‐Mediated Chromosomal Truncation for Generating Engineered Minichromosomes in Maize

Nathan C. Swyers1, Jon P. Cody1, Morgan E. McCaw1, Nathaniel D. Graham1, Changzeng Zhao1, Robert T. Gaeta1, James A. Birchler1

1 Division of Biological Sciences, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20031
Online Posting Date:  September, 2016
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Minichromosomes have been generated in maize using telomere‐mediated truncation. Telomere DNA, because of its repetitive nature, can be difficult to manipulate. The protocols in this unit describe two methods for generating the telomere DNA required for the initiation of telomere‐mediated truncation. The resulting DNA can then be used with truncation cassettes for introduction into maize via transformation. © 2016 by John Wiley & Sons, Inc.

Keywords: minichromosomes; telomere‐mediated truncation; telomere cloning

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Table of Contents

  • Introduction
  • Basic Protocol 1: In‐Gel Ligation of Telomere DNA into Target Plasmid
  • Alternate Protocol 1: Production of Telomere Fragments Via PCR
  • Support Protocol 1: Delivery of Transgene with Telomere Arrays Into Plants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: In‐Gel Ligation of Telomere DNA into Target Plasmid

  • LB plates (see recipe) containing 100 mg/ml spectinomycin (e.g, Sigma‐Aldrich, cat. no. S4014)
  • Plasmid pWY82 (e.g., Addgene, cat. no. 65721)
  • 2× yeast extract tryptone (YT) medium (see recipe)
  • QIAprep Spin Miniprep Kit (e.g., Qiagen, cat. no. 27104)
  • Nuclease‐free water
  • Restriction endonucleases compatible with pWY82 and target plasmid
  • 6× DNA Gel Loading Dye (e.g., Thermo Fisher Scientific, cat. no. R0611)
  • GeneRuler 1 kb DNA Ladder (e.g., Thermo Fisher Scientific, cat. no. SM0311)
  • Agarose (e.g., Sigma‐Aldrich, cat. no. A9539)
  • Ethidium bromide (e.g., Sigma‐Aldrich, cat. no. E7637)
  • Target plasmid for transformation
  • Low‐melting‐point agarose (e.g., Invitrogen, cat. no. 16520‐050)
  • 1× TAE (see recipe)
  • Antarctic Phosphatase (e.g., New England BioLabs, cat. no. M0289S)
  • T4 DNA Ligase and Ligase Reaction Buffer (e.g., New England BioLabs, cat. no. M0202S)
  • ElectroMax Stbl4 cells (e.g., Thermo Fisher Scientific, cat. no. 11635‐018)
  • SOC medium (e.g., Thermo Fisher Scientific, cat. no. 15544‐034)
  • LB plates (see recipe) containing appropriate antibiotics for transformation vector
  • Radiolabeled nucleotide: (TTTAGGG) 10
  • Agrobacterium
  • Sterile applicator stick or inoculating loop
  • Variable temperature incubator
  • Variable temperature laboratory shaker
  • 250‐ml baffled culture flasks
  • NanoDrop spectrophotometer
  • 1.7‐ml microcentrifuge tubes
  • Vacuum concentrator
  • UV transilluminator
  • Scalpel
  • 70°C and 37°C water bath
  • 15‐ml culture tubes
  • Nitrocellulose membrane
  • Additional reagents and equipment for gel electrophoresis (Green and Sambrook, ), electroporation (Potter and Heller, ), colony hybridization (Sambrook et al., ), colony PCR (Woodman, ), DNA sequencing (Shendure et al., ), and Southern hybridization (Hoopes, )

Alternate Protocol 1: Production of Telomere Fragments Via PCR

  • Telomere DNA to be amplified
  • Nuclease‐free water
  • LongAmp Taq DNA Polymerase and 5× Reaction Buffer (e.g., New England Biolabs, cat. no. M0323S)
  • Telomere primers (500 ng/µl):
  • Forward primer: 5′‐(TTTAGGG) 10‐3′
  • Reverse primer: 5′‐(CCCTAAA) 10‐3′
  • 10 mM dNTP mix
  • Wizard SV Gel and PCR Clean‐Up System (e.g., Promega, cat no. A9281)
  • Thermal cycler
  • Additional equipment and reagents for gel electrophoresis (Green and Sambrook, )
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Literature Cited

Literature Cited
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